Preparation Of Monoclonal Antibody Against Sarafloxacin And Its Application In The ELISA

Fluoroquinolones are bactericidal antibiot-ics that have been increasingly used in veterinary medicine to treat microbial infections. They are con-geners of nalidixie acid and exert their bactericidal effects by inhibiting DNA gyrase within susceptible bacteria. Recently ,sarafloxacin became the first fluoroquinolone to be approved by the U.S.Food and Drug Administration(FDA) for use in food animals,and the only approved use is in day-old broiler chickens to control early mortality associated with Escherichia coli. But in recent years, the use of SARA is being increased, and the reports about the adverse effect and resistance of SARA are becoming more and more, so its latent serious adverse effect is concerned by public gradually. Furthermore, as the veterinary drugs and additives of animal feed, the SARA is widely used for animals, as a result, the possibility of SARA residues in animal edible tissue is becoming higher. Not only the side effect can endanger the human's health directly, but also a lower density of residues of SARA may make the sensitive germ to SARA bear the SARA and this endanger the human's health indirectly.To preserve the usefulness of this valuable class of antibiotics, steps must be taken to minimize the poten-tial for developing resistant pathogens. These steps include monitoring test samples of bacteria to measure the emergence of any resistant pathogens and detecting extra-lable use in other major food-producing animal species by screening for fluoroquinolone residues. Con-ventional methods for detection of fluoroquinolones include a spectrofluorometric assay and high-performance liquid chroma-tography (HPLC) assays. These methods are labor intensive and require expensive equipment, therefore, they cannot be used for routine screening of large numbers of sample.Immunoassay screening methods have been successfully developed as alternatives to the convertional microbiological or chemical methods for detecting pes-ticides(insecticides and herbicides), drug residues and undesirable natural products. In contrast to microbiological assays, immunoassays are highly specific,and unlike conventional chemical assays, they require minimal sample preparation procedures.According to the basic principle of immunology, the complete antigen of the SARA were prepared and the monoclonal antibody (McAb) of the anti- SARA(SARA-McAb) were obtained. An indirect competitive inhibition enzyme-linked immunosorbent assay (ic-ELISA) was established to detect the SARA residues in food animal edible tissues, and it will lay foundations for further studying of the fast test kits for detecting SARA in animal derived food.The SARA was conjugated directly to bovine serum albumin (BSA) and ovalbumin (OVA) by EDC method. The conjugates SARA-BSA and SARA-OVA were analyzed by nondenaturing gel electrophoresis and UV absorbance method, the molecule conjugate ratio of SARA to BSA and OVA were 2︰1 and 3︰1 respectively. Splenocytes from mice immunized with BSA-SARA were fused with SP2/0 myeloma cells. Three hybridomas of stable secreting SARA-McAb were selected and were named 3B8 and 4H8, after the OVA-SARA as coating antigen was used to recognize the antibodies produced against SARA. The SARA-McAb of 4H8 was used to establish the ELISA method after the character of the three hybridomas being analyzed. The number of chromosome of the hybridoma cell line was from 82 to 95. The subclasses of McAb was IgG1; the molecular was 143KD; the ELISA titers of ascites was 1︰100×214; the effective concentration of McAb was 3.77mg/ml; the affinity constant was 4.28×1010M-1; the cross-reactivities with norfloxacin,ciprofloxacin,enrofloxacin,fleroxacin and gatifloxacin were 76.8%,80.3%,92.8%,84.9%and 104.5%. Respectively, the non-fluoroquinolones were tested and there were no cross-reaction between them.According to competitive principle, an ic-ELISA method was established for the quantitative detection of SARA and its optimized reaction condition as follow:The OVA- SARA was diluted to microtiter plates at a concentration of 1μg/ml and incubated 1h at 37℃. Plates were washed three times with PBST for 30 second per times, and 150μl 1% gelatin was added to each well to eliminate nonspecific binding by blocking the plastic surface where protein was not bound. After 30 minutes of incubation at 37℃.Plates were washed three times with PBST, and 1︰22000 SARA-McAb and varying concentrations of standard SARA (50μl/well) were added. After 2h of incubation at 37℃, Plates were washed three times with PBST, and 100μl/well goat anti-mouse IgG-HRP (1︰5000) was added and incubated for 1h at 37℃. Plates were washed four times, and 100μl/well OPD substrata solution was added, followed by the addition of stopping solution(2M H2SO4) after 10 minutes of incubation in the dark at 37℃. Absorbance at 490nm was determined by an enzyme immunoassay reader. Percent inhibiting(b/b0×100%) was calculated from the absorbance obtained in the presence(b) and absence(b0) of SARA in standard. A linear dose-response standard curve was prepared by plotting log [SARA] versus percent inhibiting. The regression equation of standard curve was y = -24.566x + 94.216,The correlation coefficient R2=0.9979,the lower limit detection was 1.55ng/ml, the average recovery rate of standard was 100.49% and the coefficients of variation was 6.97%.Collect the chicken organizational samples that were sold on the market, each sample was divided into three groups ,adding the standard SARA 200ng/g, 100ng/g and 50ng/g to each group respectively. After the fore treatment, the stock solution was used to ic-ELISA directly, and the results was as follow:The spiking concentration of standard SARA was from 50ng/g to 200ng/g. The average recoveries of ic-ELISA was 84.52% and the coefficients of variation was 4.60% in the cordis of chicken, respectively; The average recoveries of ic-ELISA was 83.93% and the coefficients of variation was 6.33% in the muscle of chicken, respectively; The average recoveries of ic-ELISA was 82.62% and the coefficients of variation was 4.57% in the chicken liver, respectively.Using the ic-ELISA method that had been established, the recoveries in the sample of chicken organizes was above 70%, and matched the common regulate of ELISA method.